Compounds and methods for stabilizing thrombin activity

ABSTRACT

The present invention is directed to compounds useful in stabilizing thrombin activity, thrombin compositions comprising the compounds, methods of using the compounds and methods of identifying compounds capable of stabilizing thrombin activity. The compounds are preferably isolated peptides comprising or interacting with the gamma loop of thrombin.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which is submittedconcomitantly with this application via EFS-Web in ASCII format and ishereby incorporated by reference in its entirety. Said ASCII copy,created on Oct. 7, 2013, is named “sequencelisting” and is 4 kilobytesin size.

FIELD OF THE INVENTION

Provided herein are compounds, compositions and formulations comprisingsame and methods useful for stabilizing thrombin activity and extendingthrombin's shelf-life. In particular, disclosed herein are isolatedpeptides comprising the amino acid sequence of the thrombin gamma loopand isolated peptides that are capable of interacting with the gammaloop of the thrombin, compositions, formulations, and methods of usetherefore to stabilize thrombin activity in a liquid thrombinformulation. Further provided is a method to identify compounds capableof stabilizing thrombin activity.

BACKGROUND

Thrombin is a serine protease which serves as an active component inseveral hemostasis products. For example, fibrin sealants typicallycomprise a fibrinogen component and a thrombin component. When bothcomponents are mixed (e.g. when applied to a bleeding wound or surgicalincision) thrombin cleaves fibrinogen and a fibrin polymer is formed.Concentrated purified thrombin in liquid form displays a reduction inactivity during prolonged storage, mostly as a result of autolysis.

Hemostatic formulations containing liquid thrombin have special handlingrequirements in order to maintain thrombin's biologic activity andprevent autolytic degradation. For example, a liquid thrombinformulation requires refrigeration or the addition of proteaseinhibitors to maintain shelf-life stability. In the clinic,refrigeration is not always feasible, and promiscuous proteaseinhibitors may adversely affect the activity of thrombin.

Thrombin may be made into a lyophilized medical preparation, which isused after dissolving at the time of use. However, liquid preparationsare advantageous as compared with the lyophilized preparations in thatthey can be easily administered without the additional step ofdissolving in a solvent prior to use.

Other known compositions and methods for stabilizing thrombin areunsatisfactory and include the following: inclusion of variousnon-specific components (e.g. bulk carrier proteins such as albumins,different stabilizing sugars, general protease inhibitors etc.);formulation of the thrombin with inhibitors of thrombin activity, whichalthough may be efficient, may also inactivate or inhibit the thrombinduring use thereby reducing its effectiveness. In order to avoid orreduce inhibition, in use, it may be needed prior to use to dilute theinhibitor and therefore the thrombin. Formulation of a low dosethrombin, necessitates administration of larger amounts of theformulation.

International Patent Application Publication No. WO2008157304 disclosesmethods for stabilizing thrombin solutions with a preservative selectedfrom benzyl alcohol or chlorobutanol and sucrose. Additional PatentPublications provide compositions comprising thrombin and non-specificinhibitors, and therefore, cannot effectively counter thethrombin-thrombin autolysis effect. For example, U.S. Pat. No. 4,409,334discloses a stabilized thrombin preparation in solid or dissolved formcomprising thrombin and as a stabilizer serum albumin together with atleast one protease inhibitor which does not inhibit thrombin itself andat least one hexyligand chelate former.

European Patent No. EP0277096 B1 provides a stable thrombin compositioncontaining purified thrombin, a polyol, and a buffer which containseither acetate or phosphate ions, wherein the preparation has a pH ofabout 5.0 to about 6.0.

European Patent No. EP 0478827 B1 provides a stable thrombin compositionwhich includes a mixture of three stabilizers: HEPES-buffer, thiomersal,gelatin obtained by partial hydrolysis of collagen, and optionallyPolybrene.

U.S. Pat. No. 7,351,561 discloses a stable thrombin preparationcomprising thrombin and benzamidine or p-aminobenzamidine as stabilizer,and further including calcium chloride or sodium chloride as stabilizer,at least one buffer substance, and at least one of histidine, mannitol,sodium succinate, sodium lactate or arginine.

US Patent Application Publication No. 20090136474 (U.S. Pat. No.8,394,372) provides a stabilized serine protease composition whichcomprises a serine protease; a reversible inhibitor of said serineprotease (e.g. benzamidine, N,N-diethylethylenediamine,aminobenzamidine); and a stabilizing agent (e.g. 3-(N-morpholino)propanesulfonic acid).

Non-patent literature describing various aspects of thrombin include:Pozzi N, et al., (2011) “Rigidification of the autolysis loop enhancesNa(+) binding to thrombin” (Biophys Chem. 159(1):6-13); Marino, F.(2010) “Engineering thrombin for selective specificity toward protein Cand PAR1” (J Biol. Chem. 285(25):19145-52); Bah A, et al., (2009)“Stabilization of the E* form turns thrombin into an anticoagulant” (JBiol. Chem. 24; 284(30):20034-40); Yang L (2004) “Heparin-activatedantithrombin interacts with the autolysis loop of target coagulationproteases” (Blood. 104(6):1753-9); and Rydel T J, et al (1994)“Crystallographic structure of human gamma-thrombin” (J Biol. Chem.269(35):22000-6).

Therefore, there remains a need for specific compounds useful tostabilize thrombin from autolytic degradation while retaining itsbiological activity. Preferably, the compounds may be used with aconcentrated liquid thrombin formulation.

SUMMARY OF THE INVENTION

Provided herein is a compound which has the exceptional ability tostabilize thrombin activity. The compound is capable of stabilizingactivity of thrombin in a liquid formulation and is useful in extendingthe shelf-life of thrombin. Without wishing to be bound to theory, thecompound inhibits, fully or partially, thrombin autolysis, whilepreserving thrombin activity toward its heterologous substrates,including fibrinogen.

One advantage is that the stabilized thrombin comprising the compoundcan be used directly for activating fibrinogen. The stabilized thrombincan be used without dilution and/or without removing the compound fromthe solution.

The compound is further beneficial in that it is potent and can be usedin low concentrations, and thus, is readily diluted upon addition of thestabilized thrombin formulation to a substrate. Further provided is acomposition or a formulation comprising the compound, methods of usingthe compound and methods of identifying such compound.

In one aspect, provided herein is a compound capable of stabilizing theactivity of thrombin in a liquid formulation, wherein the compound isselected from the group consisting of an isolated peptide which includesthe amino acid sequence of the thrombin gamma loop, a derivative or saltthereof; and a thrombin gamma loop interacting molecule, a derivative orsalt thereof. In some embodiments, the amino acid sequence of thethrombin gamma loop includes an amino acid sequence KETWTANVGK set forthin SEQ ID NO:1.

In some embodiments, the compound is an isolated peptide comprising thegamma loop peptide, a derivative or salt of such peptide or of suchderivative.

In some embodiments, the compound is an isolated gamma loop peptide, aderivative or salt of such peptide or of such derivative.

In some embodiments, the peptide is linear or cyclized.

In some embodiments, the isolated gamma loop peptide is linear.

In some embodiments, the linear isolated gamma loop peptide includes anamino acid sequence set forth in SEQ ID NO:1, a derivative or a saltthereof.

In preferred embodiments, the linear isolated gamma loop peptide,derivative or a salt thereof has an amino acid sequence set forth in SEQID NO:1.

In some embodiments, the isolated peptide comprises the thrombin gammaloop sequence and one or more amino acids flanking the gamma loop.

In some embodiments the isolated peptide includes 3-4 amino acids ateach terminus of the peptide. The amino acids can be, for example, theamino acids that are naturally adjacent to the thrombin gamma loop aminoacid sequence. In various embodiments, the isolated peptide has an aminoacid sequence set forth in SEQ ID NO:2 (GNLKETWTANVGKGQPS).

In some embodiments, the isolated gamma loop peptide is cyclized.

In some embodiments, the cyclized isolated gamma loop peptide includesan amino acid sequence set forth in SEQ ID NO:1. In preferredembodiments, the cyclized isolated peptide includes a cysteine residueat both termini of the gamma loop amino acid sequence and has an aminoacid sequence set forth in SEQ ID NO:3 (CKETWTANVGKC).

In some embodiments, the compound is a thrombin gamma loop interactingmolecule, a derivative or salt thereof.

In various embodiments, the interacting molecule is selected from anisolated interacting peptide or derivative thereof, an isolated antibodyor antibody fragment thereof, a nucleotide aptamer or a peptide aptamer;or a salt of such a molecule. In preferred embodiments, the interactingmolecule is an isolated interacting peptide, or a derivative or saltthereof.

In various embodiments, the isolated interacting peptide is an isolatedthrombin peptide which does not include the gamma loop peptide ofthrombin, the amino acid sequence of which is derived from the linearthrombin amino acid sequence or from a non-linear, surface facing,folded amino acid sequence of thrombin. For example, the amino acidsequences of the thrombin peptides can be based on the three dimensionalstructure of thrombin and not necessarily include a primary sequence ofthrombin polypeptide. In some embodiments, the isolated thrombin peptideincludes an amino acid sequence of SEQ ID NOS:7, 9, 10 or 11. In someembodiments, the isolated thrombin peptide has an amino acid sequence ofSEQ ID NOS:7, 9, 10 or 11. In one embodiment, the isolated thrombinpeptide, derivative or salt thereof is a linear peptide. In anotherembodiment, the isolated thrombin peptide derivative or salt thereof iscyclic. In some embodiments, the cyclic thrombin peptide has an aminoacid sequence set forth in any one of SEQ ID NOS:7, 9, 10 or 11, and acysteine residue at each of the amino and carboxy termini.

In various embodiments, the isolated interacting peptide, derivative orsalt thereof is obtained from a random peptide library. In someembodiments, the random isolated interacting peptide includes an aminoacid sequence of SEQ ID NOS:12 or 13. In one embodiment, the randomisolated interacting peptide is a linear peptide. In some embodiments,the random isolated interacting peptide has an amino acid sequence ofSEQ ID NOS:12 or 13. In another embodiment, the random isolatedinteracting peptide is a cyclic peptide. In some embodiments, the cyclicpeptide includes an amino acid sequence set forth in any one of SEQ IDNOS:12 or 13, and a cysteine residue at each of the amino and carboxytermini.

In a second aspect, provided herein is a composition comprising acompound capable of stabilizing the activity of thrombin in a liquidthrombin formulation, wherein the compound is selected from an isolatedpeptide comprising the amino acid sequence of the thrombin gamma loop, aderivative or salt thereof; and a thrombin gamma loop interactingmolecule, a derivative or salt thereof. In some embodiments, thecompound is present in the composition in an amount effective tostabilize thrombin activity, for example, to inhibit thrombin autolysiswithout significantly compromising thrombin biological activity; and apharmacologically acceptable excipient. In some embodiments, thethrombin biological activity comprises cleavage of fibrinogen to fibrin.

In another aspect, provided herein is a thrombin formulation comprisingthrombin, a compound capable of stabilizing the activity of thrombin inthe formulation, wherein the compound is selected from an isolatedpeptide comprising the amino acid sequence of the thrombin gamma loop, aderivative or salt thereof; and a thrombin gamma loop interactingmolecule, a derivative or salt thereof; and a pharmacologicallyacceptable excipient.

In some embodiments, the formulation or composition includes thrombinwith a thrombin activity of about 1 IU/ml to 10,000 IU/ml, of about 10IU/ml to 5,000 IU/ml or preferably of about 10 IU/ml to 1,000 IU/ml.

In preferred embodiments of the formulation or composition, the compoundis an isolated peptide, a derivative or salt of such peptide or of suchderivative.

In some embodiments, the interacting molecule is a thrombin derivedpeptide or is obtained from a random peptide library.

In some embodiments, the compound is a thrombin derived peptide.

In some embodiments, the compound, e.g. peptide, is present in thecomposition or formulation at a concentration of about 0.01 mM to about20 mM, about 0.01 mM to about 1 mM, about 0.1 mM to about 1 mM, about0.1 mM to about 0.5 mM, or about 0.5 mM.

Within one embodiment, the compound, composition or formulation arecontained in a sealed container having a label affixed to an exteriorsurface thereof. In some embodiments, the formulation or composition isprepared for use as a fibrin sealant component.

In another aspect, the invention features a kit containing an effectiveamount of a compound disclosed herein, and directions for using thecompound to stabilize thrombin in a liquid formulation. In preferredembodiments, the compound is an isolated peptide, a derivative or saltof such peptide or of such derivative.

In yet another aspect, provided is a method of stabilizing thrombinactivity, comprising contacting the thrombin with an isolated peptidecomprising the amino acid sequence of the thrombin gamma loop, aderivative or salt thereof or with a molecule that interacts with thegamma loop of the thrombin, in an amount effective to stabilize thrombinactivity. In some embodiments, stabilizing thrombin activity comprisesinhibiting thrombin autolysis without significantly compromising itsbiological activity.

In yet another aspect, provided is a method of stabilizing thrombinactivity, comprising contacting the thrombin with a compound or thecomposition disclosed herein.

In another aspect provided herein is a method for screening for acompound capable of stabilizing the activity of thrombin in liquid form,comprising

-   -   a) providing an isolated peptide comprising the amino acid        sequence of the thrombin gamma loop;    -   b) providing a set of test compounds;    -   c) contacting the isolated peptide of (a) with the set of test        compounds of (b); and    -   d) Identifying one or more test compounds, which bind to the        peptide of a).        whereby the binding indicates a potential compound for use in        stabilizing thrombin activity.

In some embodiments, the method further includes the step of isolatingthe one or more compounds identified in step (d).

In another aspect provided herein is a method for screening for acompound capable of stabilizing the activity of thrombin in liquid form,comprising

-   -   a) providing an isolated peptide comprising the amino acid        sequence of the thrombin gamma loop bound to a solid phase;    -   b) providing a gamma loop sequence;    -   c) providing a set of test labelled compounds;    -   d) contacting the isolated peptide of (a) with the set of test        compounds of (c) in the presence and absence of the gamma loop        sequence of b);    -   e) measuring the level of labelled test compound bound to the        solid phase in the presence and absence of the gamma loop        sequence whereby a substantially unaltered label level in the        presence and absence of the gamma loop sequence is indicative        that the compound is a candidate for stabilizing thrombin        activity.

The labelling can be fluorescent, radioactive labelling or any otherlabelling known in the art.

In some embodiments, the method further includes the step of testing theone or more identified or candidate compound(s) for their effect instabilizing the activity of thrombin in liquid form.

In some embodiments, the method further includes the step of testing theone or more identified or candidate compounds for A- their effect instabilizing the activity of thrombin in liquid form and B- minimal or noinhibition of the peptide on activity towards heterologous substratese.g. fibrinogen.

In some embodiments of the method, the thrombin gamma loop includes anamino acid sequence set forth in SEQ ID NO:1. In various embodiments,the set of test compounds is obtained from a random peptide library, achemical compound library, an antibody library, a peptide phage displaylibrary, an aptamer library, and the like. In some embodiments, thecompound is safe and non-immunogenic.

In some embodiments, the compound is an isolated peptide. In someembodiments, the peptide is synthesized chemically or recombinantly. Invarious embodiments, provided is a recombinant peptide encoded by anisolated nucleic acid sequence. In some embodiments, the peptidecomprises an amino acid sequence set forth in any one of SEQ ID NOS:1-3, 7 or 9-13.

Provided herein is an isolated nucleic acid sequence encoding thepeptide disclosed herein and a vector comprising the nucleic acidsequence encoding such peptide, operatively linked to a promoterelement. Further provided is a host cell comprising such vector. The DNAsequence can be extrapolated using the standard genetic code (forexample, Lehninger, A. “Principles of Biochemistry”).

These and other aspects and embodiments of the invention will becomeevident upon reference to the following detailed description of theinvention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an illustration of the prothrombin molecule. Thesequence which is cleaved during prothrombin activation is shown aslight gray silhouette. The mature alpha-thrombin sequence is shown inblack. The gamma loop peptide sequence is silhouetted in the thrombinprimary sequence, and is shown separately below the prothrombin molecule(indicated by an arrow, SEQ ID NO:1). SEQ ID NO:2 is a peptidecomprising the gamma loop sequence (marked with brackets) with three andfour amino acids flanking on the N- and C-termini, respectively.

FIG. 2A shows the inhibitory effect that arginine*HCl (“arginine”) hason thrombin. Arginine addition to liquid thrombin results in a 50%reduction in thrombin activity at 0.5% (w/v) and >95% reduction inthrombin activity at 2% (w/v).

FIG. 2B shows the stabilization level of concentrated thrombin obtainedwith different concentrations of arginine at 37° C.

FIG. 2C shows an increase in thrombin stabilization level (% activityremaining over time) obtained by either addition of increasing amountsof a gamma loop peptide to liquid thrombin (1000 IU/ml) or addition of aconstant (3% w/v) arginine concentration to liquid thrombin (1000 IU/ml)or without any addition of peptide or inhibitor. Measured was theremaining activity of 1000 IU/ml thrombin after 72 and 144 hours at 37°C.

FIG. 2D shows % inhibition of thrombin activity (measured at 10 IU/mlthrombin) by increasing concentration of the gamma peptide.

FIGS. 3A-3F are graphs showing stabilization or destabilization level ofthrombin by peptides disclosed herein. Thrombin at 1000 IU/ml wasincubated with 0.5 mM of the various peptides in vials, as indicated.The remaining activity in the individual vials was measured afterincubation at 37° C. for 0, 3 and 7 days. Benzamidine, a direct activesite inhibitor of thrombin was used as a control at 0.5 mM. A controlgroup without any peptide or inhibitor additives is also included. Theresults are divided by groups:

FIG. 3A is a graph showing stabilization level of thrombin (as shown bymeasuring % of remaining activity) with random peptides capable ofbinding the gamma loop.

Rnd316 (SEQ IDN NO:12) is listed in the Fig. as Random1.

Rnd155 (SEQ IDN NO:13) is listed in the Fig. as Random2.

FIG. 3B is a graph showing stabilization of liquid thrombin (as shown bymeasuring % of remaining activity) using gamma peptides, both a linearpeptide (SEQ ID NO: 2) and a peptide cyclized via intramolecular S—Sbonding (“CS”; SEQ ID NO:3).

FIG. 3C is a graph showing stabilization level of thrombin activity (as% of remaining activity) using cyclized mutant gamma loop peptide (SEQID NO: 4 [AL-cyc_E03N], SEQ ID NO: 5 [AL-cyc_N08Y] and SEQ ID NO: 6[AL-cyc_G10L]).

FIG. 3D shows destabilization level of thrombin activity (as % ofremaining activity) using the thrombin derived peptide Thr-111 (SEQ IDNO:8).

FIG. 3E is a graph showing stabilization level of thrombin activity (as% of remaining activity) using thrombin derived peptide Thr-069 (SEQ IDNO:7).

FIG. 3F is a graph showing stabilization level of thrombin activity (as% of remaining activity) using linearized peptides in which Cysteineresidues have been replaced to Serine (SEQ ID NO: 9 [Thr_(—)031_CS], SEQID NO: 10 [Thr_(—)032_CS] and SEQ ID NO: 11 [Thr_(—)136_CS]).

FIGS. 4A and 4B show levels of thrombin inhibition (%) by gammaloop/binding peptides (the inhibition can be calculated from the % ofremaining activity shown in the graph): thrombin derived peptides (Thr031 CS [SEQ ID NO:9], Thr 032 CS [SEQ ID NO:10]); cyclic gamma peptide[SEQ ID NO:3]; Peptide Rnd 316 [SEQ ID NO:12], and linear gamma peptide[SEQ ID NO:1].

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, in part, upon the finding thatcompounds, in particular, certain isolated peptides which include thethrombin gamma loop sequence or interact with the thrombin gamma loopare capable of stabilizing thrombin activity in a thrombin liquidformulation.

“Stabilizing thrombin activity” refers to, for example, reducingthrombin autolytic activity.

“Stabilizing thrombin activity” may also refer to maintaining thrombinactivity when stored for more than one day, e.g. at room temperature asan aqueous thrombin solution e.g. a concentrated thrombin solution,without significantly compromising thrombin's biological activitytowards heterologous substrates, including the activity of conversion offibrinogen to fibrin.

“Room temperature” is meant to include temperature of about 20° C. toabout 28° C., or 22° C. to about 26° C.

The term “stabilizing” means, for example, maintaining the thrombinactivity within the thrombin liquid formulation at a level of about 80%to about 100% (e.g. about 90 to 100%) compared to the initial thrombinactivity.

The term “initial thrombin activity” refers, for example, to theactivity of thrombin towards fibrinogen measured in a thrombin liquidformulation immediately after thawing a frozen thrombin formulation,immediately after reconstituting thrombin powder and/or before storageof liquid thrombin under conditions that allow self degradation (e.g.more than one month storage at 2-8° C.; more than 1 day at roomtemperature e.g. at concentrations of 10 IU/ml to 5,000 IU/ml thrombinor more) thrombin.

It was found that a linear or cyclic (i.e. intramolecular S—S bonds)gamma loop peptide or linear or cyclic peptide which contains theconsecutive amino acid sequence of the thrombin gamma loop; peptidesknown to interact with the gamma loop of thrombin; and randomly selectedpeptide that show a binding interaction with the gamma loop stabilizeliquid thrombin.

It was found that peptides comprising the gamma loop sequence (SEQ IDNO:2 and SEQ ID NO:3), whether linear or cyclic, displayed an efficientstabilization of thrombin activity. The cyclic gamma peptide (SEQ IDNO:3) showed only ˜7% inhibition of thrombin at 0.5 mM, and the lineargamma peptide (SEQ ID NO:1) exhibited about 20% inhibition

It was found that stabilization of thrombin with cyclic peptides mutatedin residues E, N or G of the gamma loop was inefficient (SEQ ID NOS: 4,5, and 6, respectively).

Two random peptides (SEQ ID NOS:12 and 13), selected from a randomlibrary on the basis of their initial binding to the gamma loop, yieldedstabilization effect of thrombin.

Binding of a molecule e.g. a peptide to the gamma loop, appears to be agood predictor of their stabilizing potential.

Two thrombin derived peptides [Thr 031 CS (SEQ ID NO:9)], Thr 032 CS(SEQ ID NO:10)] which are capable of interacting with the gamma looppeptide, showed stabilization effect on thrombin and exhibited a minorinhibitory effect on thrombin at the same concentrations (e.g. 0.5 mMpeptide: 13-14% inhibition).

It was found that peptides with sequence set forth in SEQ ID NOS: 2, 3,7, 9, 10, 11, 12 and 13 were capable of stabilizing liquid aqueousthrombin.

The stabilized thrombin, comprising the molecules/peptides foundaccording to the invention, can be used directly for activatingfibrinogen e.g. without dilution and/or removal of themolecules/peptides.

For stability testing, 1000 IU/ml thrombin following storage with orwithout peptides can be used and activity testing can be carried out asdescribed herein. Dilutions (1:100) can be carried out before measuringthe activity.

For inhibition testing, the clotting activity of 10 IU/ml thrombin canbe measured in the presence or absence of peptides (without theirdilution).

Thrombin activity towards fibrinogen can be assessed by measuringthrombin clotting activity. The clotting activity can be measureddirectly, for example, by the modified, European Pharmacopeia Assay(0903/1997) procedure and/or indirectly, such as measuring migrationlength on a slanted surface (or drop test model), or by any other methodknown in the art.

Provided herein are compounds e.g. isolated peptides that include athrombin gamma loop sequence or that interact with the thrombin gammaloop. Further provided herein is a method of identifying compoundscapable of stabilizing thrombin activity in an aqueous liquid thrombinformulation.

Provided herein are compounds capable of stabilizing the activity ofthrombin in a liquid thrombin formulation. The compounds are selectedfrom the group consisting of isolated peptides comprising the amino acidsequence of the thrombin gamma loop, derivatives or salts thereof; andthrombin gamma loop interacting molecules, and derivatives or saltsthereof.

A peptide comprising the amino acid sequence of the thrombin gamma loopand a thrombin gamma loop interacting molecule are different form theintact alpha thrombin.

Amino Acids

Amino acids and peptide sequences are commonly abbreviated as shownbelow, in Table A.

TABLE A Abbreviation, systematic name and formulae of common amino acidsSymbols/ abbreviations Name 3 ltr 1 ltr Systematic name Formula AlanineAla A 2-Aminopropanoic acid CH3—CH(NH2)—COOH Arginine Arg R 2-Amino-5-H2N—C(═NH)—NH— guanidinopentanoic acid [CH2]3—CH(NH2)— COOH AsparagineAsn N 2-Amino-3- H2N—CO—CH2— carbamoylpropanoic acid CH(NH2)—COOHAspartic acid Asp D 2-Aminobutanedioic acid HOOC—CH2—CH(NH2)— COOHCysteine Cys C 2-Amino-3- HS—CH2—CH(NH2)— mercaptopropanoic acid COOHGlutamine Gln Q 2-Amino-4- H2N—CO—[CH2]2— carbamoylbutanoic acidCH(NH2)—COOH Glutamic acid Glu E 2-Aminopentanedioic acid HOOC—[CH2]2—CH(NH2)—COOH Glycine Gly G Aminoethanoic acid CH2(NH2)—COOH HistidineHis H 2-Amino-3-(1H-imidazol-4- |       yl)propanoic acidNH—CH═N—CH═C—CH2— CH(NH2)—COOH Isoleucine Ile I 2-Amino-3- C2H5—CH(CH3)—methylpentanoic CH(NH2)—COOH Leucine Leu L 2-Amino-4- (CH3)2CH—CH2—methylpentanoic acid CH(NH2)—COOH Lysine Lys K 2,6-Diaminohexanoic acidH2N—[CH2]4—CH(NH2)— COOH Methionine Met M 2-Amino-4- CH3—S—[CH2]2—(methylthio)butanoic CH(NH2)—COOH Phenylalanine Phe F 2-Amino-3-C6H5—CH2—CH(NH2)— phenylpropanoic acid COOH Proline Pro PPyrrolidine-2-carboxylic |      acid NH—CH2)3—CH—COOH Serine Ser S2-Amino-3- HO—CH2—CH(NH2)— hydroxypropanoic acid COOH Threonine Thr T2-Amino-3- CH3—CH(OH)— hydroxybutanoic acid CH(NH2)—COOH Tryptophan TrpW 2-Amino-3-(lH-indol-3-yl)- |      Ph—NH— propanoic acidCH═C—CH2—CH(NH2)— COOH Tyrosine Tyr Y 2-Amino-3-(4-hydroxy HO—p-Ph—CH2—phenyl)-propanoic acid CH(NH2)—COOH Valine Val V 2-Amino-3-(CH3)2CH—CH(NH2)— methylbutanoic acid COOH

In one embodiment, an amino acid analog sequence is used whereby atleast one amino acid in the isolated peptide is substituted with ananalog or bio-similar amino-acid (conservative substitution), as knownin the art.

The amino acids can be in L-form, D-form, or their derivatives (e.g.pseudo amino acid, functionalized amino acid (e.g. fluorinated aminoacid . . . etc.), beta amino acid, gamma amino acid . . . etc.).

Thrombin

Thrombin is a serine protease which results from the cleavage ofprothrombin (Factor II), a zymogen, by another serine protease (FactorXa). Human thrombin is a 295 amino acid protein composed of twopolypeptide chains joined by a

The zymogen prothrombin (shown in FIG. 1) is cleaved at residue 271,removing the entire N-terminal 271 amino acids. An additionalintramolecular cleavage by Factor Xa at residue 320 yields the activealpha thrombin molecule which is a 295 amino acid polypeptide (human)composed of a heavy and light chain held together via a single S—S bond(Krishnaswamy J, (2013) “The transition of prothrombin to thrombin”. JThromb Haemost. June; 11 Suppl 1:265-76.) Thrombin, being a serineprotease, can initiate its own degradation (“autolysis”) by cleavingother thrombin molecules at the beta (residue 382 and 394) or gamma(residue 443 and residue 474) sites, yielding beta- and gamma-thrombin,respectively.

Neither of these loops contain a classic thrombin recognition site, noris this cleavage specific to a certain residue within the loops. Rather,these loops are both flexible and exposed and are cleaved for lack of aproper substrate and especially at high thrombin concentration (see forexample, Chang, J Y. Biochem. J. (1986) 240:797-802, “The structures andproteolytic specificities of autolysed human thrombin”; Rydel T J, etal., J Biol Chem. 1994, 269(35):22000-6. Crystallographic structure ofhuman gamma-thrombin”; Pozzi N, et al., Biophys Chem. 2011, 159(1):6-13“Rigidification of the autolysis loop enhances Na(+) binding tothrombin”). The inactivation of thrombin in-vivo does not proceed viathis mechanism (autolysis) but rather via a specific interaction(bridged by heparin) with the serine protease inhibitor (SERPIN),anti-thrombin III (ATIII).

The interaction of thrombin (and several other homologous serineproteases such as Factor X and even protein C) with ATIII is mediatedvia the gamma loop (see, for example, Yang, L., Blood. 2004,104(6):1753-9, “Heparin-activated antithrombin interacts with theautolysis loop of target coagulation proteases”; and Marino, F, J BiolChem. 2010, 285(25):19145-52. “Engineering thrombin for selectivespecificity toward protein C and PAR1”).

Human and non-human thrombin can be used within the present invention.Thrombin is used medically e.g. as a hemostatic agent and as a componentof tissue adhesive.

In one aspect, provided herein is a thrombin formulation comprising: a)thrombin; and b) a compound capable of stabilizing thrombin activity,the compound selected from the group consisting of: an isolated peptidecomprising the amino acid sequence of the thrombin gamma loop, aderivative or salt thereof; and a thrombin gamma loop interactingmolecule, a derivative or salt thereof; and a pharmacologicallyacceptable excipient.

For long-term storage, the formulation, comprising the thrombin and thecompound, is aliquoted into sterile vials, ampoules, or othercontainers, which are then sealed. In one embodiment, a seal thatpermits removal of the stabilized thrombin composition with a syringethrough the seal is used. The container can be labeled according tostandard practice in the pharmaceutical or medical device field.

In one embodiment of the invention, the container is provided in a kitwith a second container containing a scaffold, such a gelatin orcollagen based matrix. In another embodiment, the container is providedin a kit with a second container comprising a fibrinogen comprisingcomponent. The kit may further comprise an application device, such as asprayer, syringe, or the like and/or a diluent and/or instructions foruse.

In use, the stabilized thrombin formulation can be used directly fromthe container or can be further diluted to the desired concentration,generally the thrombin activity in the formulation is from about 1 IU/mlto about 10,000 IU/ml, typically about 10 IU/ml to 5,000 IU/ml, or 10IU/ml to 1,000 IU/ml although the actual concentration will bedetermined by the user (e.g. physician, nurse, medic) i.e. according tothe needs of the individual patient and on the severity of bleeding. Thestabilized thrombin can be applied to bleeding tissue to achievehemostasis, per se or may be used in combination with a scaffold ormatrix, for example, an absorbable scaffold or matrix. The stabilizedthrombin formulation can also be used as a component of a tissueadhesive, fibrin sealant or fibrin glue. These and other known in theart uses of thrombin are contemplated for the disclosed stabilizedthrombin. Numerous uses of fibrin glue in various fields have beenreported, including use as a sealant e.g. for sealing leaks, hemostaticagent/stop bleeding, adhesion prevention, to enhance healing, forjoining structures, in a variety of open and laparoscopic surgeries.

Preferred hemostatic scaffolds are natural or genetically engineeredabsorbable polymers or synthetic absorbable polymers, or mixturesthereof. Examples of natural or genetically engineered absorbablepolymers are proteins, polysaccharides and combinations thereof.Proteins include, prothrombin, thrombin, fibrinogen, fibrin,fibronectin, heparinase, Factor X/Xa, Factor VII/VIIa, Factor IX/IXa,Factor XI/XIa, Factor XII/XIIa, tissue factor, batroxobin, ancrod,ecarin, von Willebrand Factor, collagen, elastin, albumin, gelatin,platelet surface glycoproteins, vasopressin, vasopressin analogs,epinephrine, selectin, procoagulant venom, plasminogen activatorinhibitor, platelet activating agents, synthetic peptides havinghemostatic activity, and/or combinations thereof. Polysaccharidesinclude, without limitation, cellulose, alkyl cellulose, e.g.methylcellulose, alkylhydroxyalkyl cellulose, hydroxyalkyl cellulose,cellulose sulfate, salts of carboxymethyl cellulose, carboxymethylcellulose, carboxyethyl cellulose, chitin, carboxymethyl chitin,hyaluronic acid, salts of hyaluronic acid, alginate, alginic acid,propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan,pectin, pullulan, xanthan, chondroitin, chondroitin sulfates,carboxymethyl dextran, carboxymethyl chitosan, chitosan, heparin,heparin sulfate, heparan, heparan sulfate, dermatan sulfate, keratansulfate, carrageenans, chitosan, starch, amylose, amylopectin,polyN-glucosamine, polymannuronic acid, polyglucuronic acid, andderivatives of any of the above. Examples of synthetic absorbablepolymers are aliphatic polyester polymers, copolymers, and/orcombinations thereof.

The prothrombin/thrombin molecule and the gamma loop chain sequence areillustrated in FIG. 1.

DEFINITIONS

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, steps or components but do not preclude the addition of one ormore additional features, steps, components or groups thereof.

When a numerical value is preceded by the term “about”, the term “about”is intended to indicate +/−10%.

As used herein, the term “peptide” is used broadly to mean an isolatedcompound of about 5 to about 100 consecutive amino acids, or analogs ofamino acids. Included within the definition of peptide are, for example,peptides containing one or more analogs of an amino acid (including, forexample, unnatural amino acids, peptoids, etc.), peptides withsubstituted linkages, as well as other modifications known in the art,both naturally occurring and non-naturally occurring (e.g. synthetic).Thus, synthetic peptides, cyclized, branched peptides and the like, areincluded within the definition. Non-limiting lengths of peptidessuitable for use in the present invention includes peptides of 5 to 100residues (amino acids and/or analogs) in length (or any integertherebetween), 5 to 20 residues in length, 6 to 75 residues in length,10 to 25 residues in length, 21 to 75 residues in length, 75 to 100residues in length. Typically, peptides useful in this invention canhave a maximum length suitable for the intended application. Preferably,the peptide is between about 5 and 30 residues in length e.g. betweenabout 5 and 30 consecutive amino acid residues; for example, about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 consecutive amino acid residues, preferably about10 to 17 or 10 to 15 residues in length.

Furthermore, a peptide as described herein, for example syntheticpeptides, may include additional molecules such as labels or tracers,linkers, or other chemical moieties (e.g. biotin, dyes) covalentlyattached thereto or non-covalently associated therewith. Such moietiesmay further enhance interaction of the peptides with the compound e.g.thrombin gamma loop peptide and/or aid in detection or quantification ofstabilized thrombin.

The term peptides also includes derivatives of the amino acid sequencesof the invention having one or more substitution, addition and/ordeletion, including one or more non-naturally occurring amino acid.Preferably, derivatives exhibit at least about 50% identity to any wildtype or reference sequence, preferably at least about 70% identity, morepreferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to any wild type orreference sequence described herein. Peptide derivatives can includemodifications to the native sequence, such as deletions, additions andsubstitutions (generally conservative in nature), so long as the peptidemaintains the desired activity e.g. stabilization of thrombin. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through synthesis or mutations of hoststhat produce the proteins or errors due to PCR amplification. Furtherencompassed herein are pharmaceutically acceptable salts of peptides andthe derivatives of such salts.

By “gamma loop peptide” is meant a peptide of ten (10) consecutive aminoacid sequence set forth in SEQ ID NO:1, specifically the sequenceKETWTANVGK (LYS-GLU-THR-TRP-THR-ALA-ASN-VAL-GLY-LYS). Without wishing tobe bound to theory, the sequence of the thrombin gamma loop ishomologous to and corresponding to residues 145-150 in bovinechymotrypsin according to the classic numbering system of this proteinfamily and has been shown using X-ray crystallography to maintain ageneral exposed loop structure. Sequences homologous to the thrombingamma loop can be contemplated as derivatives of the thrombin gammaloop.

“Thrombin” or “thrombin polypeptide” is a mammalian serine proteasewhich is part of the blood coagulation cascade and converts fibrinogeninto insoluble strands of fibrin, as well as catalyzes othercoagulation-related reactions. In humans, prothrombin is encoded by theF2 gene, and the resulting polypeptide is proteolytically cleaved in thecoagulation cascade to form thrombin. Thrombin serves, inter alia, as anactive component in several hemostasis products. For example, fibrinsealants typically comprise a fibrinogen component and a thrombincomponent. When both components are mixed (e.g. when applied to ableeding wound) thrombin cleaves fibrinogen and a fibrin polymer isformed.

One skilled in the art will recognize that the peptides disclosed hereinmay be synthesized as derivatives of the peptides, including “peptidemimetics”. A peptide mimetic or “peptidomimetic”, is a molecule that isnot completely peptidic in nature, yet mimics the biological activity ofthe peptide upon which it is structurally based. Such peptidomimeticsinclude peptide-like molecules containing non-naturally occurring aminoacids. A peptidomimetic can include one or more amino acid analogs andcan be a peptide-like molecule which contains, for example, an amidebond isostere such as a retro-inverso modification; reduced amide bond;methylenethioether or methylenesulfoxide bond; methylene ether bond;ethylene bond; thioamide bond; trans-olefin or fluoroolefin bond;1,5-disubstituted tetrazole ring; ketomethylene or fluoroketomethylenebond or another amide isostere. The terms also include moleculescomprising one or more N-substituted glycine residues (a “peptoid”) andother synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos.5,831,005; 5,877,278; and 5,977,301; Nguyen et al. (2000) Chem. Biol.7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA89(20):9367-9371 for descriptions of peptoids). One skilled in the artunderstands that these and other peptidomimetics are encompassed withinthe meaning of the term “peptidomimetic” as used herein.

The amino acid sequence of a peptide is written according to theconventional notation, with an amino group (NH2) at the N-terminalappearing on the left hand of the sequence and carboxyl group (COOH) atthe C-terminal appearing on the right hand thereof.

The peptides disclosed herein may form a physiologically acceptable saltby conventional salt formation reaction. Such salts can include saltswith inorganic acids such as hydrochloric acid, sulfuric acid andphosphoric acid; salts with organic acids such as lactic acid, tartaricacid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid,oleic acid and palmitic acid; salts with hydroxides and carbonates ofalkali metals and alkali earth metals such as sodium, potassium, calciumand aluminum; and salts with amines such as triethylamine, benzylamine,diethanolamine, t-butylamine, dicyclohexylamine and arginine.

Both inter- and intra-chain disulfide bonds may be formed and thepeptide formed resulting from the formation of such disulfide bonds areencompassed by the present invention.

In one embodiment, the peptides disclosed herein are chemicallysynthesized. In other embodiments, the peptides disclosed herein areproduced in-vivo or ex-vivo by expression of recombinant DNA inprokaryotic or eukaryotic host cells.

In other embodiments, the peptides disclosed herein are produced in-vivoor ex-vivo by expression of a vector comprising the nucleic acidsequence encoding the compound disclosed herein in prokaryotic oreukaryotic host cells.

The terms “isolated polynucleotide”, “isolated nucleic acid sequence”and “an isolated nucleic acid molecule” are used herein interchangeably.An isolated “polynucleotide” can include both double- andsingle-stranded sequences and refers to, but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from viral, prokaryotic oreukaryotic mRNA, genomic RNA and DNA sequences from viral (e.g. RNA andDNA viruses and retroviruses), prokaryotic DNA or eukaryotic (e.g.mammalian) DNA, and synthetic DNA sequences. The term also encompassessequences that include known base analogs of DNA and RNA, and includesmodifications such as deletions, additions and substitutions (generallyconservative in nature), to the native sequence. Modifications ofpolynucleotides may have any number of effects including, for example,facilitating expression of the peptide in a host cell. Typically, thepolynucleotide encodes peptides of at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or even moreamino acids.

A “polynucleotide coding sequence” or a sequence that “encodes” aselected polypeptide, is a nucleic acid molecule that is transcribed (inthe case of DNA) and translated (in the case of mRNA) into a polypeptidein vivo when placed under the control of appropriate regulatorysequences (or “control elements”). The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxyl) terminus. A transcriptiontermination sequence may be located 3′ to the coding sequence. Typical“control elements,” include, but are not limited to, transcriptionregulators, such as promoters, transcription enhancer elements,transcription termination signals, and polyadenylation sequences; andtranslation regulators, such as sequences for optimization of initiationof translation, e.g. Shine-Dalgarno (ribosome binding site) sequences,Kozak sequences (i.e., sequences for the optimization of translation,located, for example, 5′ to the coding sequence), leader sequences(heterologous or native), translation initiation codon (e.g. ATG), andtranslation termination sequences. Promoters can include induciblepromoters (where expression of a polynucleotide sequence operably linkedto the promoter is induced by an analyte, cofactor, regulatory protein,etc.), repressible promoters (where expression of a polynucleotidesequence operably linked to the promoter is included by an analyte,cofactor, regulatory protein, etc.), and constitutive promoters.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

A “recombinant” nucleic acid molecule as used herein to describe anucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin ormanipulation: (1) is not associated with all or a portion of thepolynucleotide with which it is associated in nature; and/or (2) islinked to a polynucleotide other than that to which it is linked innature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. “Recombinant host cells,” “host cells,” “cells,” “celllines,” “cell cultures,” and other such terms denoting prokaryoticmicroorganisms or eukaryotic cell lines cultured as unicellularentities, are used interchangeably, and refer to cells which can be, orhave been, used as recipients for constructs, vectors or other transferDNA, and include the progeny of the original cell which has beentransfected. It is understood that the progeny of a single parental cellmay not necessarily be completely identical in morphology or in genomicor total DNA complement to the original parent, due to accidental ordeliberate mutation. Progeny of the parental cell which are sufficientlysimilar to the parent to be characterized by the relevant property, suchas the presence of a nucleotide sequence encoding a desired peptide, areincluded in the progeny intended by this definition, and are covered bythe above terms.

By “isolated” is meant, when referring to a polynucleotide or a peptide,that the indicated molecule or compound is separate and discrete fromthe whole organism with which the molecule or compound is found innature or, when the polynucleotide or peptide is not found in nature, issufficiently free of other biological macromolecules so that thepolynucleotide or peptide can be used for its intended purpose.

As used herein, a molecule e.g. a peptide is said to “interact” with or“bind” to another peptide or protein (e.g. a thrombin gamma loopinteracting molecule with thrombin) if it associates with the peptide orprotein via non-covalent binding forces, for example van der Waals andelectrostatic forces. A molecule e.g. a peptide is said to “interactpreferentially” with a particular domain in a protein (e.g. the thrombingamma loop) if it associates with greater affinity and/or greaterspecificity to the particular domain than to another domain in theprotein. In some embodiments, the molecule e.g. peptide bindspreferentially to the gamma loop of thrombin. It is to be understoodthat a preferential interaction does not necessarily require interactionbetween specific amino acid residues and/or motifs of each peptide.

“Thrombin activity” and “thrombin biological activity” is meant toinclude thrombin mediated conversion of heterologous substrates,including proteins e.g. fibrinogen into fibrin, as well as theconversion of Factor VIII to Factor VIIIa, XI to XIa, XIII to XIIIa, andFactor V to Va. A “heterologous substrate” is a substrate, preferably aprotein substrate, other than thrombin. In some embodiments, thethrombin activity refers to conversion of fibrinogen into fibrin. Theterm “without significantly compromising thrombin's (biological)activity” refers to retaining thrombin activity towards fibrinogen at alevel of at least 60%, at least 70% and preferably at least 80%, or atleast 90% or more e.g. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%compared to uninhibited/unstabilized thrombin and/or compared to theinitial thrombin activity.

As used herein the terms “autolysis” or “auto degradation” refer to theunfavorable molecular degradation of thrombin into an inactive orpartially active form.

A preferred compound as disclosed herein, is a compound capable ofstabilizing thrombin activity, for example, by reducing thrombinautolysis without significantly compromising thrombin activity e.g.towards fibrinogen.

In one embodiment, the stabilized aqueous liquid thrombin formulation isstable for more than one month storage at a temperature of 2 to 8° C.;for 72 hours at 37° C.; and/or for 144 hours at 37° C.

In some embodiments the compound inhibits autolysis of thrombin by about60% to about 100% or about 60% to about 95%, preferably by about 70% toabout 90%, and retains about 60% to about 100% or about 60% to about95%, about 70% to about 90%, preferably about 80% to about 95% thrombinbiological activity, e.g. after one month storage at a temperature of 2to 8° C. in liquid form; after 72 hours at about 37° C.; after 144 hoursat about 37° C.

The term “affinity” refers to the strength of binding and can beexpressed quantitatively as a dissociation constant (K_(d)). A moleculee.g. a peptide disclosed herein can interact with the gamma loop ofthrombin with at least 2 fold greater affinity, more preferably at least5 fold greater affinity and even more preferably at least 10, 20, 30, 40or 50-fold greater affinity than it interacts with another domain ofthrombin. Binding affinity (i.e., K_(d)) can be determined usingstandard techniques.

The term “an effective amount” refers to the amount of a compounddisclosed herein required to stabilize thrombin while substantiallyretaining thrombin activity e.g. towards fibrinogen (e.g. conversion offibrinogen to fibrin). The effective amount of a compound used topractice the present invention for stabilization of thrombin may varydepending upon the concentration of thrombin in acomposition/formulation. Such amount is referred to as an “effectiveamount”.

The “pharmaceutically acceptable” or “pharmacologically acceptable”carriers, solvents, diluents, excipients, and vehicles generally referto inert, non-toxic solid or liquid fillers, diluents or encapsulatingmaterial not reacting with the active ingredients of the compositionsdisclosed herein. Acceptable excipients include, without limitation,saline; acetic acid or acetate; calcium, sodium and chloride ions;mannitol; albumin; or combination thereof.

The term “contacting” is used herein in its broadest sense and refers toany type of combining action. Contacting includes, but is not limitedto, mixing, admixing and/or adding.

Peptide Synthesis

Peptides disclosed herein may be synthesized according to methods knownin the art, including, but not limited to synthetic (e.g. synthesizingthe peptide chemically from individual amino acids) and recombinantmethods (e.g. synthesizing DNA encoding the peptide and using the DNA toproduce recombinant peptide).

Chemical synthesis of the peptide: a peptide disclosed herein and DNAencoding the peptide may be chemically synthesized by methods known inthe art. Suitable methods for synthesizing the peptide are described byStuart and Young (1984), “Solid Phase Peptide Synthesis”, Solid PhasePeptide Synthesis, Methods Enzymol., Second Edition, Pierce ChemicalCompany, 289, Academic Press, Inc., NY (1997). For example, a solidphase synthesis method or a liquid phase synthesis method may be used.The solid phase synthesis is usually carried out by protecting aminogroups with appropriate protecting groups. For example, either Boc(tert-butoxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl), or acombination thereof may be used. In one example, a peptide disclosedherein is synthesized by following the steps: 1) an amino acid residuecorresponding to the C-terminal of the peptide to be produced is bondedto a solid phase material insoluble to a reaction solvent via an a-COOHgroup of the amino acid or such solid phase material is purchased; 2) inthe direction towards the N-terminal of the peptide, a correspondingamino acid or peptide fragment is bonded by condensation to the aminoacid of step 1) after protecting other functional groups such as ana-amino group of the corresponding amino acid or peptide fragment otherthan an a-COOH group; 3) a protecting group of an amino group forming apeptide bond such as an a-amino group is removed from the bonded aminoacid or peptide fragment; 4) steps 2) and 3) are repeated to elongate apeptide chain in order to form a peptide chain corresponding to thedesired peptide; 5) detach the produced peptide chain from the solidphase material and remove the protecting groups from the protectedfunctional groups; and 6) purify the peptide, thereby to obtain thedesired peptide.

Solid phase materials, as well as solvents and a condensing agents, arewell known in the art.

Chemical synthesis and expression of DNA: The DNA encoding a peptidedisclosed herein may be replicated and used to express recombinantpeptide following insertion into a wide variety of host cells in a widevariety of cloning and expression vectors. The host may be prokaryoticor eukaryotic. The DNA may be chemically synthesized. Suitable methodsfor synthesizing DNA and cloning vectors (e.g. for use in mammalian,insect or plant cells, bacteria, phage and yeast) are available. Therecombinant peptide, which can be expressed in the form of a fusionprotein, is purified by methods known in the art.

Compounds Useful in Practicing the Present Invention

Provided herein are compounds and methods for stabilization of thrombinactivity in liquid thrombin formulation, wherein stabilizing thethrombin activity refers, for example, to reducing or preventingautolytic activity without significantly compromising the thrombin'sbiological activity. The compounds are selected from the groupconsisting of an isolated peptide comprising the amino acid sequence ofthe thrombin gamma loop peptide, a derivative or salt thereof; or athrombin gamma loop interacting molecule which may be an isolatedinteracting peptide, an isolated antibody or antibody fragment thereof,a nucleotide aptamer and a peptide aptamer, a derivative or a salt ofsuch a molecule.

Molecules that interact with the gamma loop of thrombin may beidentified in a screen and may be tested for their ability to stabilizethrombin activity, using, for example, the methods disclosed herein. Insome embodiments, the interacting molecule is an isolated peptide, apeptidomimetic of such peptide, or a salt of such peptides. Examples ofinteracting peptides are provided herein, for example in SEQ ID NOS: 7,and 9-13.

Peptides may be linear, branched or cyclized. For example, peptidesrepresented by SEQ ID NO:1 and 2 are linear, and the peptide representedby SEQ ID NO:3 is cyclic.

In some embodiments, the interacting molecule is an isolated antibody, afragment of such antibody, or a salt of such antibody. The term“antibody” refers to IgG, IgM, IgD, IgA, and IgE antibody, inter alia,and includes polyclonal antibodies and monoclonal antibodies. In oneembodiment the antibody is directed towards, was raised against, and/orrecognizes the thrombin gamma loop. This term refers to whole antibodiesor fragments of antibodies comprising an antigen-binding domain, e.g.antibodies without the Fc portion, single chain antibodies,miniantibodies, fragments consisting of essentially only the variable,antigen-binding domain of the antibody, etc. The term also encompassesantibody derivatives such as antibody fragments which retain the abilityto selectively bind with their antigen or receptor and are exemplifiedas follows, inter alia:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule which can be produced by digestion ofwhole antibody with the enzyme papain to yield a light chain and aportion of the heavy chain;

(2) (Fab′)₂ of the antibody is a dimer of two Fab fragments heldtogether by disulfide bonds, that can be obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction.

(3) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(4) Single chain antibody (SCA), defined as a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

In some embodiments, the interacting molecule is an aptamer or a salt ofsuch aptamer. Aptamers are RNA and/or DNA single-strand or double-strandoligonucleotides, which bind to a target protein and do not generallyexhibit non-specific effects. Aptamers can be modified for stability orother desired qualities in accordance with any nucleic acidmodifications known to one of skill in the art. Modifications to anaptamer can be introduced anywhere in the molecule, such as the 5′ or 3′termini, or at any internally defined modification site. Thioaptamersare aptamers which contain sulfur modifications at specificinternucleoside phosphoryl sites, and may possess enhanced stability,nuclease resistance, target affinity and/or selectivity. Examples ofthioaptamers include phosphoromonothioate (S-ODN) and phosphorodithioate(S2-ODN) oligodeoxy thioaptamers. Further information on aptamers andthioaptamers can be found in U.S. Pat. Nos. 5,218,088 and 6,423,493.

Details of the exemplary compounds useful in practicing the invention,are provided in the Examples, hereinbelow and in the sequence listing,incorporated herewith.

Methods of Screening

Provided herein are methods of screening for compounds capable ofstabilizing thrombin activity. Accordingly, provided is a method forscreening for a compound capable of stabilizing the activity of thrombinin a liquid thrombin formulation, comprising:

-   -   a) providing an isolated peptide comprising the amino acid        sequence of the thrombin gamma loop;    -   b) providing a set of test compounds;    -   c) contacting the isolated peptide of (a) with the set of test        compounds of (b); and    -   d) identifying one or more compounds, which bind to the peptide;        whereby the binding indicates a potential use of the compound in        stabilizing thrombin activity.

In some embodiments, the method further includes the step of isolatingthe one or more compounds identified in step d) and/or of testing theone or more compounds identified in step d) for its ability to stabilizethrombin activity.

While the following examples demonstrate certain embodiments of theinvention, they are not to be interpreted as limiting the scope of theinvention, but rather as contributing to a complete description of theinvention.

EXAMPLES Example 1 Thrombin Activity Assay

Aqueous liquid thrombin, in its purified and concentrated form 1000international units IU/ml may undergo autolysis at room temperaturecausing a significant loss of activity e.g. towards a heterologoussubstrate. E.g. thrombin activity towards a heterologous substrate isreduced when a liquid thrombin formulation is incubated at roomtemperature for prolonged periods of time (e.g. 72 to 144 hours), interalia, due to autolytic degradation. The decrease of thrombin activitytowards a heterologous substrate in aqueous liquid solution can beassessed by measuring thrombin activity after prolonged periods of timeand under permissive temperature (e.g. 37° C.). In the followingexperiments, the effect of a peptide on the stability of thrombin wasstudied under different conditions.

Aqueous liquid purified and concentrated thrombin (1000 IU/ml;equivalent to about 10 μM) was aliquoted and placed in a 37° C.incubator for 3 days (72 hours), 7 days (168 hours) and 14 days (336hours). Prior to incubation, thrombin samples were spiked with indicatedamounts of different tested peptides or with a control thrombininhibitor: benzamidine or arginine *HCl (“arginine”). Specific peptides,their concentrations, and thrombin inhibitors used are indicated in eachExample. Following the incubation period, the samples were frozen at−80° C. until testing the thrombin activity. Just before thrombinactivity assay testing, all the samples were thawed and 100-fold dilutedinto a dilution buffer (0.4% tri-sodium citrate di-hydrate, 0.9% sodiumchloride and 1% BSA, pH=7.5) to bring the thrombin concentration in thesample to that within the specifications of the assay (4-10 IU/ml) andto dilute the tested peptides in the sample to a negligibleconcentration.

Thrombin activity was assessed by clotting time measurements usingSTart4 Coagulation Instrument (Diagnostica Stago, Asnieres sur Seine,France). The assay is a modification of the European Pharmacopoeia Assayprocedure, 1997, 0903, p. 858. Briefly, a calibration curve was preparedby mixing thrombin standard with a fibrinogen solution of 0.1%fibrinogen content (Enzyme Research Laboratories, 1N, USA). Thrombinconcentration in the different tested samples was then calculated fromthe calibration curve by their clotting time (the concentration wasextrapolated from the calibration curve).

For stability testing of 10001 U/ml thrombin following storage with orwithout peptides, testing was carried out as described above. Dilutions(1:100) were carried out before measuring the activity.

For inhibition testing, the clotting activity of 10 IU/ml thrombin wasmeasured in the presence or absence of peptides.

Example 2 The Effect of Peptides Comprising the Amino Acid Sequence ofthe Thrombin Gamma Loop on Thrombin Stabilization

The prothrombin sequence which is cleaved during thrombin formation isshown as light gray silhouette in FIG. 1. The mature alpha-thrombinpolypeptide sequence is shown in black. The peptide sequence utilizedherein is silhouetted in the thrombin primary sequence, and shownseparately below the molecule (indicated by the arrow). The gamma looppeptide sequence is shown (SEQ ID NO:1), per se, and bracketed within alonger peptide which includes amino acids flanking on both N- andC-termini (SEQ ID NO:2; GNLKETWTANVGKGQPS;GLY-ASN-LEU-LYS-GLU-THR-TRP-THR-ALA-ASN-VAL-GLY-LYS-GLY-GLN-PRO-SER).SEQ ID NO:1 was cyclized by synthesizing the peptide with terminalcysteine residues (SEQ ID NO:3; CKETWTANVGKC;CYS-LYS-GLU-THR-TRP-THR-ALA-ASN-VAL-GLY-LYS-CYS). These peptides weresynthesized by standard methods.

The gamma loop peptide with the flanking amino acids (SEQ ID NO:2), andcyclic peptide (SEQ ID NO:3) were used to test for inhibition ofthrombin autolytic activity. Arginine *HCl (“arginine”), a thrombinknown active site inhibitor, was used as a control. FIG. 2A shows theinhibitory effect that arginine has on thrombin activity (measured using10 IU/ml thrombin) at different arginine concentrations. FIG. 2B showsthe measured % of thrombin after incubation for up to 48 hours at 37° C.with different concentrations of arginine (as shown in the graph).Concentrated thrombin was used for shorter times (up to 48 hours) inorder to rapidly obtain a working range for arginine. Arginine displaysa dose-dependent effect on thrombin stability correlating to itsinhibitory effect seen in FIG. 2A.

FIG. 2C shows thrombin remaining activity after incubation with eitherincreasing amounts of gamma loop peptide (SEQ ID NO:2) or with aconstant concentration of (3% w/v) arginine. The assay was based onmeasuring the remaining activity of 1000 IU/ml thrombin after 72 and 144hours at 37° C.

At 0.1 mM of peptide there was already stabilization detected after 144hours, and at 0.2 mM it was already evident after 72 hours. FIG. 2Dshows inhibition of thrombin activity (measured at 10 IU/ml thrombin)with increasing concentration of peptide. At peptide concentration of0.2 mM, thrombin activity is not significantly affected

Results: A range of 0.5-5% (w/v) arginine maintained thrombin activityfollowing storage (FIG. 2B). However, the presence of argininecompromised thrombin biological activity (see FIG. 2A). Even at 0.5%(w/v) arginine, about 50% (w/v) of the thrombin activity was inhibitedand >95% of thrombin activity is inhibited at a concentration of 2%(w/v) (as assayed by its ability to cleave fibrinogen; FIG. 2A). Basedon the effective arginine concentration, 3% (w/v) arginine was comparedto a 0.5 mM concentration of the gamma loop peptide (SEQ ID NO:2; FIG.2C). Surprisingly, when the peptide was used at the stabilizingconcentration of 0.5 mM, thrombin activity remained high (about 80%remaining activity, see FIG. 2D). In this same experiment, 3% (w/v)arginine was not more effective at maintaining thrombin activity at 72hours, and only marginally so at 144 hours. This is significant as 3%(w/v) arginine concentration can be extrapolated (based on FIG. 2A) toinhibit thrombin almost entirely.

At a 0.5 mM concentration of peptide, an increase in thrombin stabilitywas observed (FIG. 2C) with a concomitant reduction of thrombin activitytowards fibrinogen of only 20% (FIG. 2D). Without wishing to be bound totheory, the gamma loop peptide appears to be, at least partially, anallosteric inhibitor of thrombin degradation.

Example 3 Screening for Mutant Gamma Loop Peptides

A screen was carried out to identify gamma loop mutants that may showimproved binding to thrombin. This was carried out with fluorescentthrombin on an array of gamma loop peptide mutants. Three such cyclizedpeptides with the highest binding efficiency were tested for theircapacity to stabilize thrombin without compromising thrombin activitytowards fibrinogen (see Example 4 below).

“Gamma loop” (SEQ ID NO:1) shows the wild type sequence of the thrombingamma loop; E03N refers to the substitution of a asparagine in place ofa glutamic acid in position 3 (in SEQ ID NO:4); N08Y refers to thesubstitution of a tyrosine in place of asparagine in position 8 (in SEQID NO:5); G10L refers to the substitution of a leucine in place of aglycine in position 10 (in SEQ ID NO:6).

The amino acid sequences of the peptides are shown in Table 1 hereinbelow:

TABLE 1 Mutant gamma loop peptides SEQ Peptide Peptide ID SEQPeptide SEQ name NO: 1-letter 3-letter Gamma 1 KETWTANVLYS-GLU-THR-TRP-THR- loop GK ALA-ASN-VAL-GLY-LYS AL- 4 CKNTWTANCYS-LYS-ASN-THR-TRP-THR- cyc_E03N VGKC ALA-ASN-VAL-GLY-LYS-CYS AL- 5CKETWTAY CYS-LYS-GLU-THR-TRP-THR- cyc_N08Y VGKC ALA-TYR-VAL-GLY-LYS-CYSAL- 6 CKETWTAN CYS-LYS-GLU-THR-TRP-THR- cyc_G10L VLKCALA-ASN-VAL-LEU-LYS-CYS

Example 4 Screening for Gamma Loop Binding Peptides

Based on the results disclosed in Example 2, the gamma loop peptide wasused as a bait to find additional peptides which may positively affectthe stability of thrombin. For this purpose, a binding screen wascarried out using a fluorescent gamma loop peptide incubated withseveral peptide arrays.

A library of 1,676 15-meric peptides spanning the sequences of proteinsinteracting with the gamma loop of thrombin such as thrombin itself,anti-thrombin III (ATIII), thrombomodulin, heparin cofactor II, bovinepancreatic trypsin inhibitor (BPTI), and hirudin, and modifications ofthe thrombin gamma loop sequence were generated. Peptides weresynthesized using SPOT synthesis (Wenschuh H, et al. (2000). Coherentmembrane supports for parallel microsynthesis and screening of bioactivepeptides. Biopolymers 55:188-206) and were chemoselectively immobilizedonto functionalized glass slides as described (Panse S, et al. (2004)Profiling of generic anti-phosphopeptide antibodies and kinases withpeptide microarrays using radioactive and fluorescence-based assays. MolDivers 8:291-299.2.). Each peptide was printed in triplicates onto themicroarray. For binding studies, 100 ng of purified thrombin (Omrixbiopharmaceuticals) were directly labeled with DyLight 650 (DyLight®Microscale Antibody Labeling Kits, Thermo Scientific, #84536) anddiluted in blocking buffer (SuperBlockT20 (TBS) Blocking Buffer, ThermoScientific, #37536). The microarrays were incubated with 10 μg/mlDyLight 650-Thrombin or a Fluorescein-labeled cyclic gamma-loop derivedpeptide for one hour at 30° C. in a HS4800 microarray processing station(Tecan) (Masch A, et al., (2010) Antibody signatures defined byhigh-content peptide microarray analysis. Methods Mol Biol 669:161-172).

Microarrays were washed with 0.1% Tween-20 in 1×TBS followed by 0.05%Tween-20 in 0.1×SSC and dried in a stream of nitrogen. Each microarraywas scanned using GenePix Autoloader 4200AL (Molecular Devices, Pixelsize: 10 μm). Signal intensities were evaluated using spot recognitionsoftware Genepix Pro 7.0 analysis software (Molecular Devices). For eachpeptide, the mean signal intensity of the three triplicates wasextracted. Further evaluation and representation of results wasperformed using the statistical computing and graphics software R(Version 2.11.1, www.r-project.org).

One array included peptides based on the thrombin sequence. Any peptideisolated from this library may reflect possible regions in the thrombinprotein that may interact with the gamma loop. A second array wascomposed of random peptides of which several arbitrarily peptides showedaffinity to the gamma loop. The fluorescence for the strongest bindingcandidates (hits) was quantified on an arbitrary scale of 0-65535 (2¹⁶).Thrombin activity was assessed as described in Example 1.

The amino acid sequences of the candidate peptides identified in thescreen from the thrombin derived peptide array (Thr; SEQ ID NO:7-11) andthe random peptide array (Rnd; SEQ ID NO:12-13) are shown in Table 2herein below:

TABLE 2Binding and sequence of different gamma-loop interacting peptidesBinding Binding SEQ intensity of intensity of ID gamma loop thrombinPeptide NO: Sequence (1-letter) (arbitrary units) (100 μg/ml) Thr_069 7WCYVAGKPGDFGYCD 50553 3141 Thr_111 8 ISMLEKIYIHPRYNW 32804 60199Thr_031_CS 9 NITR S GIESQLWR S R 40787 18537 Thr_032_CS 10 S GIESQLWR SRYPHK 35117 15079 Thr_136_CS 11 RIRITDNMF S AGYKP 25278 5535Rnd_inter_316 12 LGNKKFVSGSRFVST 26628 12353 Rnd_inter_155 13SHNQRFVTYLGSKLG 19539 23646 Underlined and in bold are the Ssubstitutions.

All of these peptides (“mutant gamma loop peptides” identified inExample 3, random peptides, thrombin derived peptides, and “gamma loopbinding peptides” mentioned above) were tested for their ability tostabilize thrombin activity at a concentration of 0.5 mM as described inExample 2.

The results are shown in the figures, as follows:

FIG. 3A shows stabilization level of thrombin activity (as % ofremaining activity) using random candidate peptide binding to the gammaloop (Random peptides represented by SEQ ID NO:12 and SEQ ID NO:13).

FIG. 3B shows stabilization level of thrombin activity (as % ofremaining activity) using peptides comprising the gamma loop sequence,“linear gamma peptide” (SEQ ID NO: 2) and cyclic via intramolecular S—Sbonding, “cyclic gamma peptide” (“CS”, SEQ ID NO:3).

FIG. 3C shows stabilization level of thrombin activity (as % ofremaining activity) using gamma loop mutant peptides circularized viaintramolecular S—S bonding and displaying an enhanced binding tothrombin.

FIG. 3D shows stabilization level of thrombin activity (as % ofremaining activity) using the thrombin derived peptide Thr-111 (SEQ IDNO:8). The results show that binding of Thr-111 (SEQ ID NO:8) tothrombin increased thrombin degradation although it did not appear tobind at the active site.

FIG. 3E shows stabilization level of thrombin activity (as % ofremaining activity) using thrombin derived peptide Thr-069 (SEQ IDNO:7). The results show that Thr-069 (SEQ ID NO:7 that exhibit very weakbinding to thrombin also showed minimal effect on thrombin stability.

FIG. 3F shows stabilization levels of thrombin (as % of remainingactivity) using mutant of thrombin derived peptides in which Cysteineresidues have been replaced by Serine residues, displaying variablebinding to the gamma loop, and all of which show some reduction inbinding when thrombin is inhibited. The amino acid sequences of thethrombin peptides are based on the three dimensional structure ofthrombin and do not necessarily include a consecutive sequence ofthrombin polypeptide. All three mutants of thrombin derived peptideswere capable of stabilizing thrombin. The least effective peptide,Thr-136CS (SEQ ID NO:11), also had the weakest fluorescent signal (Table2, weakest binding to thrombin).

The data from Table 2 and FIGS. 3A-3F indicate the following:

Peptides comprising the gamma loop sequence (SEQ ID NO:2 and SEQ IDNO:3), whether linear or cyclic, displayed similar and efficientstabilization of thrombin activity (FIG. 3B).

Relatively weak thrombin interacting peptides [such as Thr-069 (SEQ IDNO:7)] and Thr-136CS [SEQ ID NO:11)] exhibited a weaker stabilizationeffect than similar peptides in the same group (FIGS. 3E and 3F).

Peptide Thr-111 (SEQ ID NO:8), having the strongest interaction withthrombin had a destabilization effect on thrombin (FIG. 3D).

Stabilization of thrombin with cyclic peptides, mutant in residues E, Nor G of the gamma loop was inefficient; (SEQ ID NOS: 4, 5, and 6,respectively) (FIG. 3C). Thus, the interaction between the gamma looppeptide and thrombin is specific, and the stabilization effect may belost if key residues are mutated, even though binding affinity may beincreased.

The two random peptides (SEQ ID NOS:12 and 13) both yielded somestabilization effect of thrombin (FIG. 3A). As was described above,these peptides were selected from a random library on the basis of theirinitial binding to the gamma loop, and this appears to be a goodpredictor of their stabilizing capacity.

Example 5 Testing Peptides for Thrombin Inhibitory Activity

Peptides that exhibited thrombin stabilization activity were tested fortheir effect on activity of thrombin toward heterologous substrates.

The assay was carried out as described in Example 1.

FIGS. 4A and B show levels of thrombin inhibition by gamma loop/thrombingamma binding peptides (the inhibition can be calculated from the % ofremaining activity shown in the graph).

FIG. 4A shows two thrombin derived peptides [Thr 031 CS (SEQ ID NO:9)],Thr 032 CS (SEQ ID NO:10)] both of which showed some stabilizationeffect on thrombin also exhibited an inhibitory effect on thrombin atthe same concentrations (e.g. 0.5 mM peptide: 13-14% inhibition). Athigher peptide concentrations (1 mM) the inhibition was greater than30%. The cyclic gamma peptide (SEQ ID NO:3) showed only ˜7% inhibitionof thrombin at 0.5 mM, while the linear gamma peptide (SEQ ID NO:1)exhibited about 20% inhibition (FIG. 2D). Both peptides displayed asimilar stabilizing effect of thrombin at this concentration. PeptideRnd 316 (SEQ ID NO:12) showed absolutely no inhibition of thrombinactivity at any concentration tested.

Benzamidine at the same concentration (0.5 mM) showed 20% inhibition inthis assay, which was higher than any of the peptides tested.

These data demonstrate that it is possible to use specific peptides forstabilizing thrombin without compromising thrombin activity (in contrastto the use of benzamidine).

FIG. 4B shows inhibition of thrombin (the inhibition can be calculatedfrom the % of remaining activity shown in the graph) by the linear (SEQID NO:1) and cyclic (SEQ ID NO:3) thrombin gamma loop peptides.

The cyclic gamma peptide (SEQ ID NO:3) showed only ˜7% inhibition at 0.5mM, and the linear gamma peptide (SEQ ID NO:1) exhibited about 20%inhibition. Both peptides displayed a similar stabilizing effect ofthrombin at this concentration.

In summary, and without wishing to be bound to theory, three categoriesof peptides are shown to stabilize thrombin:

1) A linear or cyclic (i.e. intramolecular S—S bonds) gamma loop peptideor linear or cyclic peptide which contains the consecutive amino acidsequence of the thrombin gamma loop;

2) Peptides selected from molecules known to interact with thrombin,such as thrombin itself but may also include anti-thrombin III,thrombomodulin or others, that show binding to the gamma loop;

3) Randomly selected peptides that show a binding interaction with thegamma loop.

In general, the above peptides do not inhibit thrombin at the sameconcentrations at which they stabilize it. Thrombin is active yetstable, thus, these peptides can be used to stabilize thrombin activityin the liquid formulation thereby retaining its activity towardheterologous substrates.

Although various embodiments have been described herein, manymodifications and variations to those embodiments may be implemented.Also, where materials are disclosed for certain components, othermaterials may be used. The foregoing description and following claimsare intended to cover all such modification and variations.

Any patent, publication, or other disclosure material, in whole or inpart, that is said to be incorporated by reference herein isincorporated herein only to the extent that the incorporated materialsdoes not conflict with existing definitions, statements, or otherdisclosure material set forth in this disclosure. As such, and to theextent necessary, the disclosure as explicitly set forth hereinsupersedes any conflicting material incorporated herein by reference.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the invention.Section headings are used herein to ease understanding of thespecification and should not be construed as necessarily limiting.

1. A compound capable of stabilizing the activity of thrombin in aliquid thrombin formulation, wherein the compound is selected from thegroup consisting of an isolated peptide comprising the amino acidsequence of the thrombin gamma loop, a derivative or salt thereof; and athrombin gamma loop interacting molecule, a derivative or salt thereof.2. The compound of claim 1, wherein the compound is an isolated peptidecomprising the amino acid sequence of the thrombin gamma loop, aderivative or salt thereof.
 3. The compound of claim 2, wherein theisolated peptide is linear or cyclic.
 4. The compound of claim 1,wherein the amino acid sequence of the thrombin gamma loop is set forthin SEQ ID NO:1.
 5. The compound of claim 2 or 3, wherein the isolatedpeptide comprises an amino acid sequence set forth in SEQ ID NO:2, aderivative or salt thereof.
 6. The compound of claim 2 or 3, wherein theisolated peptide comprises an amino acid sequence set forth in SEQ IDNO:3, a derivative or salt thereof.
 7. The compound of claim 2 or 3,wherein the isolated peptide consists essentially of an amino acidsequence set forth in SEQ ID NO:1, a derivative or salt thereof
 8. Thecompound of claim 2 or 3, wherein the isolated peptide consistsessentially of an amino acid sequence set forth in SEQ ID NO:2, aderivative or salt thereof.
 9. The compound of claim 2 or 3, wherein theisolated peptide consists essentially of an amino acid sequence setforth in SEQ ID NO:3, a derivative or salt thereof.
 10. The compound ofclaim 1, wherein the gamma loop interacting molecule is an isolatedinteracting peptide, a derivative or salt thereof.
 11. The compound ofclaim 10, wherein the isolated interacting peptide is a thrombinpeptide.
 12. The compound of claim 11, wherein the isolated interactingpeptide is linear or cyclic.
 13. The compound of claim 12, wherein theisolated interacting peptide comprises an amino acid sequence set forthin any one of SEQ ID NOS:7, 9, 10 or 11, a derivative or salt thereof.14. The compound of claim 12, wherein the isolated interacting peptideconsists essentially of an amino acid sequence set forth in any one ofSEQ ID NOS:7, 9, 10 or 11, a derivative or salt thereof.
 15. Thecompound of claim 10, wherein the isolated interacting peptide isobtained from a random peptide library.
 16. The compound of claim 15,wherein the isolated interacting peptide is linear or cyclic.
 17. Thecompound of claim 16, wherein the isolated interacting peptide comprisesan amino acid sequence set forth in SEQ ID NOS:12 or 13, a derivative orsalt thereof.
 18. The compound of claim 16, wherein the isolatedinteracting peptide consists essentially of an amino acid sequence setforth in SEQ ID NOS:12 or 13, a derivative or salt thereof.
 19. Apharmaceutical composition comprising the compound of claim 1; and apharmacologically acceptable excipient.
 20. A thrombin formulationcomprising thrombin, the compound of claim 1, and a pharmacologicallyacceptable excipient.
 21. The formulation of claim 20 having a thrombinactivity of about 1 IU/ml to 10,000 IU/ml.
 22. The formulation of claim20 having a thrombin activity of about 10 IU/ml to 5,000 IU/ml.
 23. Theformulation of claim 20 having a thrombin activity of about 10 IU/ml to1,000 IU/ml.
 24. The formulation of claim 20, wherein the compound ispresent at a concentration of about 0.01 mM to 1 mM.
 25. The formulationof claim 24, wherein the compound is present at a concentration of about0.1 mM to 0.5 mM.
 26. The formulation of claim 24, wherein the compoundis present at a concentration of about 0.5 mM.
 27. The formulation ofclaim 20 for use as a fibrin sealant component.
 28. The compound ofclaim 1, the composition of claim 19 or the formulation of claim 20contained in a sealed container having a label affixed to an exteriorsurface thereof.
 29. A method of stabilizing thrombin activity,comprising contacting the thrombin with the compound of claim 1 or thecomposition of claim
 19. 30. A method for screening for a compoundcapable of stabilizing the activity of thrombin in liquid form,comprising: a) providing an isolated peptide comprising the amino acidsequence of the thrombin gamma loop; b) providing a set of testcompounds; c) contacting the isolated peptide of (a) with the set ofcompounds of (b); and d) identifying one or more test compounds, whichbind to the peptide; whereby the binding indicates a potential compoundfor use in stabilizing thrombin activity.
 31. The method of claim 30,further comprising isolating the one or more compounds identified instep (d).
 32. The method of claim 30 or 31, further comprising testingthe one or more compounds identified in step (d) for their effect instabilizing the activity of thrombin in liquid form.
 33. An isolatednucleic acid sequence encoding the compound of claim
 2. 34. A vectorcomprising the nucleic acid sequence of claim 33; operatively linked toa promoter element.
 35. A host cell comprising the vector of claim 34.